The dynamic interaction of an enzyme with its substrate occurs along a well-defined catalytic pathway that consists of multiple, elementary steps. As this pathway is traversed, transient enzyme-substrate (E . S) intermediates are formed. In order to fully understand the mechanism of enzyme catalysis, it is essential that both the detailed nature of the enzyme-substrate interaction and the structures of both the enzyme and substrate in each of these intermediates be elucidated. In the past, studies of E . S intermediates have proven difficult because of their brief lifetime and low concentration. Furthermore, general methods capable of providing information about structure and interactions within the active sites of enzymes in solution have not been developed. The proposed research combines two areas of current research to overcome these problems. Sub-zero temperatures and cryosolvents will be used to accumulate intermediates in high concentrations for periods of time that permit their spectroscopic study. Resonance Raman spectroscopy will then be used to study intermediates in which either the substrate or enzyme contains a chromophoric probe to yield vibrational spectra of parts of the acitve site. These spectra are capable of providing the needed structural information. This research will focus on metalloenzymes because they often have chromophoric centers that are ideally suited for resonance Raman studies.